REPRESSOR SENSITIVE MUTATIONS IN DROSOPHILA

我们的目标是建立一个系统，可以识别和研究果蝇阻遏物敏感（rs）突变。阻遏物敏感突变是一类新的条件致死突变。为了产生rs突变，转座子P [UAS]（一种含有Gal4蛋白多个UAS结合位点的P元件）将被插入果蝇基因组的各个位置。Gal4-阻遏物融合蛋白将用于抑制与P [UAS]插入物相邻的基因的转录。rs突变的示意图如下所示。图A显示了被转录的基因的野生型等位基因。小图B显示基因的rs等位基因。与该基因相邻的是携带UAS结合位点的P元件插入物。P元件插入本身不引起突变表型。图C显示了rs突变纯合并表达Gal4-阻遏物融合蛋白的果蝇。蛋白质将结合到UAS位点并抑制相邻基因的表达，导致突变表型。通过控制Gal4阻遏蛋白的表达，可以调节突变表型表达的时间和地点。之所以需要这个系统，是因为在整个发育过程中，许多基因在多个时间点起作用。如果一个基因具有致死表型，那么在第一次需要时研究该基因要容易得多，而不是在以后的发育时间点。对于研究胚胎后发育的实验室来说，这是一个严重的障碍，迫使他们使用像镶嵌分析这样繁琐的方法。需要一个更简单的方法。阻遏物敏感突变将允许在发育期间的任何时间直接分析致死突变。我们将首先在hedgehog基因中进行rs突变。然后，我们将测试一些阻遏蛋白，以确定哪一个具有最强的阻遏活性。这种阻遏物将用于果蝇rs突变的筛选。

Our goal is to create a system that allows the identification and study of repressor sensitive (rs) mutations in the fruit fly Drosophila melanogaster. Repressor sensitive mutations are a new class of conditional lethal mutation. To make rs mutations, the transposon P[UAS] (a P element that contains multiple UAS binding sites of the Gal4 protein) will be inserted at various locations throughout the Drosophila genome. A Gal4-repressor fusion protein will be used to inhibit the transcription of genes adjacent to the P[UAS] insert. A schematic diagram of a rs mutation is shown below. Panel A shows wild type alleles of a gene being transcribed. Panel B shows a rs allele of the gene. Adjacent to the gene is a P element insert carrying UAS binding sites. The P element insertion by itself does not cause a mutant phenotype. Panel C shows a fly homozygous for a rs mutation and expressing a Gal4-repressor fusion protein. The protein will bind to the UAS sites and repress the expression of the adjacent gene, resulting in a mutant phenotype. By controlling the expression of the Gal4-repressor protein you can regulate when and where the mutant phenotype is expressed. The reason this system is needed is because many genes act at multiple time points throughout development. If a gene has a lethal phenotype, then it is much easier to study the gene the first time it is required, rather than at a later developmental time point. For labs studying post-embryonic development, this is a severe hindrance, forcing the use of cumbersome approaches like mosaic analysis. An easier approach is required. Repressor sensitive mutations will allow the direct analysis of lethal mutations at any time during development. We will initially make a rs mutation in the gene hedgehog. We will then test a number of repressor proteins to determine which one has the strongest repressor activity. This repressor will then be used in a screen for rs mutations in Drosophila.